Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast

Sister chromatid cohesion in meiosis is established by cohesin complexes, including the Rec8 subunit. During meiosis I, sister chromatid cohesion is destroyed along the chromosome arms to release connections of recombined homologous chromosomes (homologues), whereas centromeric cohesion persists until it is finally destroyed at anaphase II. In fission yeast, as in mammals, distinct cohesin complexes are used depending on the chromosomal region; Rec8 forms a complex with Rec11 (equivalent to SA3) mainly along chromosome arms, while Psc3 (equivalent to SA1 and SA2) forms a complex mainly in the vicinity of the centromeres. Here we show that separase activation and resultant Rec8 cleavage are required for meiotic chromosome segregation in fission yeast. A non-cleavable form of Rec8 blocks disjunction of homologues at meiosis I. However, displacing non-cleavable Rec8 restrictively from the chromosome arm by genetically depleting Rec11 alleviated the blockage of homologue segregation, but not of sister segregation. We propose that the segregation of homologues at meiosis I and of sisters at meiosis II requires the cleavage of Rec8 along chromosome arms and at the centromeres, respectively

Single nucleolus precursor body formation in the pronucleus of mouse zygotes and SCNT embryos.

Mammalian oocytes and zygotes have nucleoli that are transcriptionally inactive and structurally distinct from nucleoli in somatic cells. These nucleoli have been termed nucleolus precursor bodies (NPBs). Recent research has shown that NPBs are important for embryonic development, but they are only required during pronuclear formation. After fertilization, multiple small NPBs are transiently formed in male and female pronuclei and then fuse into a single large NPB in zygotes. In cloned embryos produced by somatic cell nuclear transfer (SCNT), multiple NPBs are formed and maintained in the pseudo-pronucleus, and this is considered an abnormality of the cloned embryos. Despite this difference between SCNT and normal embryos, it is unclear how the size and number of NPBs in pronuclei is determined. Here, we show that in mouse embryos, the volume of NPB materials plays a major role in the NPB scaling through a limiting component mechanism and determines whether a single or multiple NPBs will form in the pronucleus. Extra NPB- and extra MII spindle-injection experiments demonstrated that the total volume of NPBs was maintained regardless of the pronucleus number and the ratio of pronucleus/NPB is important for fusion into a single NPB. Based on these results, we examined whether extra-NPB injection rescued multiple NPB maintenance in SCNT embryos. When extra-NPBs were injected into enucleated-MII oocytes before SCNT, the number of NPBs in pseudo-pronuclei of SCNT embryos was reduced. These results indicate that multiple NPB maintenance in SCNT embryos is caused by insufficient volume of NPB

The post-anaphase SUMO pathway ensures the maintenance of centromeric cohesion through meiosis I-II transition in mammalian oocytes

The production of haploid gametes requires the maintenance of centromeric cohesion between sister chromatids through the transition between two successive meiotic divisions, meiosis I and meiosis II. One mechanism for the cohesion maintenance is shugoshin-dependent protection of centromeric cohesin at anaphase I onset [1–3]. However, how centromeric cohesion is maintained during late anaphase I and telophase I, when centromeric shugoshin is undetectable [1–3], remains largely unexplored. Here we show that the centromeric small ubiquitin-related modifier (SUMO) pathway is critical for the maintenance of centromeric cohesion during post-anaphase-I periods in mouse oocytes. SUMO2/3 and E3 ligase PIAS are enriched near centromeres during late anaphase I and telophase I. Specific perturbation of the centromeric SUMO pathway results in precocious loss of centromeric cohesin at telophase I, although shugoshin-dependent centromeric protection at anaphase I onset remains largely intact. Prevention of the SUMO perturbation during post-anaphase-I periods restores the maintenance of centromeric cohesion through the meiosis I-II transition. Thus, the post-anaphase-I centromeric SUMO pathway ensures continuous maintenance of centromeric cohesion through the meiosis I-II transition.This work was supported by research grants JSPS KAKENHI26930008 (to M.K.), 24770173/16H01226/16H06161 (to T.S.K.), MINECO BFU2017-89408-R and CyLe (to A.M.P.), and RIKEN intramural grants.Peer Reviewe

Shugoshin enables tension-generating attachment of kinetochores by loading Aurora to centromeres

Fission yeast shugoshin Sgo1 is meiosis specific and cooperates with protein phosphatase 2A to protect centromeric cohesin at meiosis I. The other shugoshin-like protein Sgo2, which requires the heterochromatin protein Swi6/HP1 for full viability, plays a crucial role for proper chromosome segregation at both mitosis and meiosis; however, the underlying mechanisms are totally elusive. We here demonstrate that, unlike Sgo1, Sgo2 is dispensable for centromeric protection of cohesin. Instead, Sgo2 interacts with Bir1/Survivin and promotes Aurora kinase complex localization to the pericentromeric region, to correct erroneous attachment of kinetochores and thereby enable tension-generating attachment. Forced localization of Bir1 to centromeres partly restored the defects of sgo2Δ. This newly identified interaction of shugoshin with Survivin is conserved between mitosis and meiosis and presumably across eukaryotes. We propose that ensuring bipolar attachment of kinetochores is the primary role of shugoshin and the role of cohesion protection might have codeveloped to facilitate this process

Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent

Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations. During the clearing process, fine morphology and fluorescent proteins were highly preserved. SeeDB2 enabled super-resolution microscopy of various tissue samples up to a depth of >100 μm, an order of magnitude deeper than previously possible under standard mounting conditions. Using this approach, we demonstrate accumulation of inhibitory synapses on spine heads in NMDA-receptor-deficient neurons. In the fly medulla, we found unexpected heterogeneity in axon bouton orientations among Mi1 neurons, a part of the motion detection circuitry. Thus, volumetric super-resolution microscopy of cleared tissues is a powerful strategy in connectomic studies at synaptic levels

Epigenetic regulation limits competence of pluripotent stem cell-derived oocytes

Recent studies have reported the differentiation of pluripotent cells into oocytes in vitro. However, the developmental competence of in vitro-generated oocytes remains low. Here, we perform a comprehensive comparison of mouse germ cell development in vitro over all culture steps versus in vivo with the goal to understand mechanisms underlying poor oocyte quality. We show that the in vitro differentiation of primordial germ cells to growing oocytes and subsequent follicle growth is critical for competence for preimplantation development. Systematic transcriptome analysis of single oocytes that were subjected to different culture steps identifies genes that are normally upregulated during oocyte growth to be susceptible for misregulation during in vitro oogenesis. Many misregulated genes are Polycomb targets. Deregulation of Polycomb repression is therefore a key cause and the earliest defect known in in vitro oocyte differentiation. Conversely, structurally normal in vitro-derived oocytes fail at zygotic genome activation and show abnormal acquisition of 5-hydroxymethylcytosine on maternal chromosomes. Our data identify epigenetic regulation at an early stage of oogenesis limiting developmental competence and suggest opportunities for future improvements.ISSN:0261-4189ISSN:1460-207